Ion channels and receptors are the basic components that shape electrical and chemical communication in the nervous system. Our long-term interest is to understand how ion channels are trafficked and regulated to perform their physiological roles. In this proposal, we focus on the highly conserved, voltage-gated Shal/Kv4 channel. Across species, these channels are localized to somato-dendritic sites, where they regulate dendritic excitability, the integration of synaptic inputs, the shape of mEPSCs, backpropagating action potentials, and long-term potentiation. Because of these important functions, animal models with decreased/mutant Shal/Kv4 channels display spatial learning defects, seizure behavior, as well as temporal lobe epilepsy. Shal/Kv4 channels have also been shown to underlie the Ito current, which is responsible for initial repolarization of the cardiac action potential. Therefore, understanding the mechanisms of Shal/Kv4 channel localization and regulation have important implications for the health and functioning of vital processes in the nervous system and heart. Using Drosophila as our model system, we propose studies to examine mechanisms underlying the somato-dendritic localization of Shal/Kv4 channels, and investigate a new role Shal/Kv4 channels play at postsynaptic sites. In Specific Aim #1, we explore two mechanisms that underlie the polarized distribution of Shal/Kv4 channels. One mechanism depends on a highly conserved di-leucine motif (LL-motif) on the C-terminus of Shal/Kv4 channels. We propose to generate a mutant of a recently identified protein, Shal Interactor of Di-Leucine Motif (SIDL) that interacts with this LL-motif, and examine how GFP-Shal/Kv4 localization is affected in vivo. This SIDL mutant will also be engineered to allow us to knock-in mutant SIDL constructs, and perform structure-function studies. We will also test whether the second mechanism involves a cytoskeletal barrier at the axon initial segment (AIS) by perturbing the AIS cytoskeleton, tracking single Shal/Kv4 channels, and analyzing how localization and mobility are affected. Specific Aim #2 is based on strong preliminary studies showing that Shal/Kv4 channels are up-regulated in response to synaptic inactivity. We will test the model that it is the homeostatic up-regulation of specific postsynaptic receptors that triggers an increase in Shal/Kv4 channel expression, for the purpose of modulating postsynaptic potentials and their homeostatic regulation. This novel regulation will add a new dimension to the role Shal/Kv4 channels play in synaptic plasticity. Using Drosophila as our model system, we combine genetic, biochemical, electrophysiological, cell and molecular biological approaches to gain unique insight into these questions that would be more difficult to address in mammalian systems. 1